Project Summary/Abstract Misfolding and aggregation of the protein ?-synuclein (?S) underlies Parkinson?s disease (PD). Current PD treatments provide only symptom relief, and significant side effects are observed. Drugs that reverse or block ?S aggregation, combined with early diagnosis, provide the best prospect for a cure that preserves the patient?s memories. To design such PD drugs and diagnostic agents, one must understand the process of aggregation within neurons and propagation to ?infect? new neurons to identify the most relevant targets. We propose to use novel protein synthesis and fluorescence techniques developed in our laboratory to structurally characterize aggregates of ?S. ?S monomers misfold, forming soluble aggregates of 2-100 ?S monomers (oligomers) as well as m long fibrils that can create insoluble, neurotoxic tangles identifiable in post-mortem analysis. Different aggregates exhibit different tendencies to form new fibrils and different levels of cytotoxicity. Determining the molecular structures of oligomers and fibrils is crucial for elucidating the aggregation pathway and directing therapies to the most toxic species. Furthermore, structural characterization of the membrane- crossing species of ?S will help to explain the spread of pathology from neuron-to-neuron. Our development of non-perturbing labeling strategies provides powerful tools to be applied to ?S characterization. Our preliminary data have shown that single residue fluorescent and crosslinking probes can be used to determine intra- and intermolecular distances in ?S aggregates. We will use our tools along with a recent solid state NMR (ssNMR) structure of fibrils to generate models of the fibrils in solution. The proposed experiments will further develop methods for synthesizing labeled versions of ?S in a manner that could be broadly applied by other researchers. We will use these to test the relevance of the ssNMR structure to fibrils formed under standard in vitro conditions as well as those formed in cells. The intra- and intermolecular distance data sets will be used in the construction of computational models of regions of ?S fibrils that are not resolved in the ssNMR structure. We will also use our semi-synthetic ?S to track structural changes upon formation of fibrils and analyze the interactions of small molecule therapeutics or diagnostics with fibrils.